Correspondence: AVIJIT DEY
S770 Pharmacognosy Magazine, Volume 13, Issue 52, October-December 2017 (Supplement 4) plant, age of leaf, types of leaf, and processing methods.[1,7,8] The catechin content is up to 30% of the dry weight, whereas the content of caffeine is up to 5% of the dry weight.[2,9] There are four major catechins in order of abundance they are epigallocatechin gallate (EGCG), EGC, epicatechin gallate (ECG), and epicatechin (EC).[9] Several therapeutic activity is reported with tea (black, green, and oolong) extracts including anticancer,[10] anti‑atherosclerosis, and as cardioprotective agents.[11] Black tea extract (BTE) is associated antiarthritic activity by altering the expression inflammatory cytokines, reducing urinary hydroxyproline, glucosamine, and histology showed that it restored the structural architecture of bones.[12] Several active bioactive constituents are associated with black teas which are theaflavins, EGCG, EGC, ECG, and EC. Theaflavin, one of the major constituent of black tea, is said to be associated with antitumorogenic activity by inhibiting proteasome activity of tumor.[13,14] Anticancer activity is also exhibited by catechin and EC another bioactive ingredient.[15] Catechins and theaflavin are also associated antioxidant and free radical scavenging activity, antimicrobial, apoptogenic activity, anti‑ischemic activity, and anti‑inflammatory activity.[16,17] Despite the increasing demand for tea/active constituents, few studies have reported their safety. Tea being the most widely drank beverage, and despite having numerous beneficial role toward health and disease, its safety evaluation during pregnancy and prenatal, postnatal developmental period needs to be monitored. Very few studies have been reported regarding tea extract consumption and its effect during pregnancy in animal models.[18] Pu‑erh black tea a highly fermented version of black tea is associated with the development of fetal toxicity at a high concentration.[5,19] In the present study, an attempt has been made to assess the effect of varying doses of BTE in pregnant and lactating rat model and to evaluate its actions on their pups through different biochemical, serum parameters, histology, and biomorphometric analysis. MATERIALS AND METHODS Chemicals Calcium kit (LABKIT, Spain), Creatinine kit (LABKIT, Spain), Di sodium hydrogen phosphate (SRL, India), Eosin (Sigma, USA), Formaldehyde solution 37%–41% w/v (Merck, India), Glacial Acetic Acid, Hematoxylin (Merck, Germany), interleukin 1 alpha (IL‑1α) rat ELISA kit, interleukin 1 beta (IL‑1 β) rat ELISA kit, interleukin 6 (IL‑6) rat ELISA kit, IL‑10 rat ELISA kit (RayBiotech, USA), Magnesium kit (LABKIT, Spain), Methanol (Merck, India), Multistrix SG Reagent Strips for Urinalysis (Siemens, India), Paraffin wax 56‑58°C (Merck, India), Picric Acid (Merck, India), Salt mixture H. M. W.(SRL, India), Sodium chloride (SRL, India), Sodium di hydrogen phosphate (SRL, India), tumor necrosis factor alpha (TNF‑α) rat ELISA kit (RayBiotech, USA), Urea kit (BEACON), and Xylene (Merck, India) were used. Animals Male (150 ± 10 g) and female (120 ± 10 g) Wistar albino rats collected from the enlisted supplier of CPCSEA, India. They were housed in polypropylene cages (421 mm × 290 mm × 190 mm) at control temperature (25 ± 2°), with light conditions (12 h light and dark cycle) and relative humidity (65% ±5%). The animals were provided with pellet diet (Ashirwad Industries, Chandigarh, India), green vegetables, gram, and water ad libitum. All animals for this experiment were kept in CPCSEA approved animal house of Maulana Azad College, Kolkata, West Bengal, India (vide F. No. – 25/250/2012‑AWD, dated 26.2.2014), and experiments described in this study were done by following the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animal (CPCSEA), Government of India. Collection of black tea Fresh black tea (C. T. C., Assam) was purchased from M/S. Subodh Brothers Pvt. Ltd., Kolkata ‑ 700 012, India. Preparation of black tea extract and treatment schedule Tea leaves were added to boiling water and kept covered for 5 min, filtered, and cooled down to 40°C.[12] This was expressed as BTE. BTE was expressed in terms of dry weight. Freshly prepared BTE was administered orally to the pregnant mother throughout their prenatal (21 days) and postnatal periods of time (21 days). Experimental design The pregnant rats were selected by pairing a proestrous female overnight with two male rats of proven fertility and determining the vaginal sperm count on the following day (9:00–9:30 a. m.) using an improved Neubauer hemocytometer. Rats with vaginal sperm counts >45 × 106/ml were selected and considered as at day 0 of pregnancy. Three groups of animals were selected for this study. Group 1 was the control group where pregnant female rats were treated with saline. Group 2 was pregnant female rats treated with BTE (50 mg BTE/kg body weight/day, p.o.), and group 3 was pregnant female rats treated with BTE (100 mg BTE/kg body weight/day, p.o.). Both the doses of BTE were administered in the pregnant mother throughout their prenatal (21 days) and postnatal periods of time (21 days). All three groups of rats were provided with pellet diet, green vegetables, gram, and drinking water ad libitum. Urine strip analysis Qualitative test of urinary glucose, bilirubin, ketone (acetoacetic acid), specific gravity, blood, pH, protein, and urobilinogen was assessed using reagent strips for urinalysis (Siemens, India). First fresh urine was collected in a clean, dry container and was mixed well. The urine was applied to different color blocks on strips and was matched with the chart present on the bottle label at the time specified and observations were recorded.[20] Urine strip analysis was done from urine of mother of three different groups at the end of lactation period following manufacturer’s instructions. Urinary marker analysis Urine was obtained (after 12 h) from all three groups of animals during prenatal period of pregnant rats on day 0, day 7, day 14, and day 20. Quantity of urinary markers such as calcium, magnesium, urea, and creatinine was obtained by performing Arsenezo III, Calmagite‑EGTA, Jaffe, and UV GLDH biochemical assays, respectively, following manufacturer’s instructions of biochemical kits (LABKIT, Spain). Histopathological examination For morphological observation by light microscopy, the kidneys from mothers of all experimental groups were collected at the end of postnatal period. The tissues were fixed in 10% neutral buffered formalin for 24 h. Tissues were then dehydrated in graded (50%–100%) ethanol followed by clearing in xylene. Paraffin (56°C–58°C) embedding was done at 58°C ± 1°C for 4 h, followed by paraffin block preparation. Tissue sections of 5 μm were cut using a rotary microtome (Weswox model MT‑1090, India). Xylene was used to deparaffinize the paraffin sections, then counterstained with hematoxylin‑eosin, and was mounted in DPX with a coverslip. Histological changes were observed with a bright field.
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